Temporal and spatial assembly of inner ear hair cell ankle link condensate through phase separation

Stereocilia are actin-based cell protrusions of inner ear hair cells and are indispensable for mechanotransduction. Ankle links connect the ankle region of developing stereocilia, playing an essential role in stereocilia development. WHRN, PDZD7, ADGRV1 and USH2A have been identified to form the so-called ankle link complex (ALC); however, the detailed mechanism underlying the temporal emergence and degeneration of ankle links remains elusive. Here we show that WHRN and PDZD7 orchestrate ADGRV1 and USH2A to assemble the ALC through liquid-liquid phase separation (LLPS). Disruption of the ALC multivalency for LLPS largely abolishes the distribution of WHRN at the ankle region of stereocilia. Interestingly, high concentration of ADGRV1 inhibits LLPS, providing a potential mechanism for ALC disassembly. Moreover, certain deafness mutations of ALC genes weaken the multivalent interactions of ALC and impair LLPS. In conclusion, our study demonstrates that LLPS mediates ALC formation, providing essential clues for understanding the pathogenesis of deafness.

6. It seems that 'Whirlin' in the pull-down, SEC-MALS and LLPS assays was Whirlin NPDZ12 but not full-length Whirlin. The authors should change the name to avoid misunderstanding.

For the sedimentation assays, it is better to quantify by densitometry, especially for those key experiments (Whirlin/Usherin at different concentrations, disruption of LLPS by adding different concentrations of VLGR1).
8. For the FRAP assay (Fig. 5H), the authors should add the original movie or the images at different time points.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): The manuscript by Wang et al provided a detailed characterization of the interaction network of the ankle-link complex formed by several USH2 proteins and demonstrated that these components can undergo liquid-liquid phase separation to form a condensed structure. This is a significant supplement to the previous findings that the tip-link densities are shown to form using similar mechanisms. More importantly, the authors showed that some mutations found in USH2 patients, although have little impact on the interactions, severely impair the LLPS properties. This provides novel insights into the potential mechanism of USH2 pathogenesis. Overall, the study is interesting and is a strong candidate for publishing in Nature Communications. I only have a few minor comments: 1. The authors should provide the Uniprot or NCBI entries of the proteins they used for obtaining recombinant proteins in the method section. Thank you for reminding us of this point. The Uniprot entries of Whirlin, PDZD7, Usherin, and VLGR1 are Q9P202, E9Q9W7, Q2QI47, and Q8VHN7, respectively. We have added the entry information in the section on molecular cloning.
2. The organization of the figures can be improved. For example: Fig. 2G is about the interaction between Whirlin and Usherin/VLGR1, and should be in Fig. 1; Fig. S2 is the pull-down assay about the interaction between PDZD7 and Usherin/VLGR1, and should be in Fig. 2. Fig. 2E&2F can be put in the supporting materials. Thank you for providing the suggestions on Figure organization. We have made the revisions according to your suggestion to make it easy to read.
3. The authors reported that the proteins were purified in a buffer containing 300 mM NaCl. Were all the assays (ITC, SEC-MALS, pull-down and LLPS) performed using the same condition? All the assays were performed using the buffer containing 300 mM NaCl. To make it clear, detailed information was added in the section of "Protein expression and purification".
A detailed characterization of the interaction between VLGR1 and PDZD7 was recently reported (Colcombet-Cazenave et al, 2022, Front Mol Biosci), and they have different conclusions. The authors should comment on this. Thank you for reminding us. The interaction between VLGR1 and PDZD7 was characterized using fluorescence titration in the above-mentioned paper. We have cited the paper and described our opinion in Result (Page 4, Line 156-159).
4. The authors used the short version of Usherin CT to test its interaction with PDZD7.
What about the WT Usherin CT? Can it bind to PDZD7 in the same manner (stoichiometry and affinity)? Thank you for raising this question. We performed the ITC assay to measure the binding affinity between the WT Usherin CT and PDZD7. As expected, the WT Usherin-CT binds to PDZD7 in a similar affinity as Usherin-CT(S) (Kd=11 vs 15 μM) (panel A below). We also used SEC-MALS to elucidate the stoichiometry of the PDZD7/Usherin-CT interaction. Usherin-CT appears as a dimer in solution. And when it is mixed with Trx-tagged PDZD7 PDZ1, the molecular size of the complex is calculated to be 77.5 kDa, which is fitted as a stoichiometry of 2:2 (panel B below).  6. It seems that 'Whirlin' in the pull-down, SEC-MALS and LLPS assays was Whirlin NPDZ12 but not full-length Whirlin. The authors should change the name to avoid misunderstanding. We are sorry for the ambiguity. For Figure 3 and the following Figures, the fragments of Whirlin NPDZ12 and PDZD7 PDZ12 were mostly used. To make it more clear and avoid misunderstanding, we have changed the names to make it easier to read. 7. For the sedimentation assays, it is better to quantify by densitometry, especially for those key experiments (Whirlin/Usherin at different concentrations, disruption of LLPS by adding different concentrations of VLGR1). We have made the quantifications of the co-sedimentation assays following the reviewers' comments. We have added a sub-panel in Figure S5A (bottom panel), showing the concentration-dependent protein condensation of WHRN/Usherin-CT at 10-20 μM (presented below). Also, Figure S7A is added to quantify the disruption of WHRN/USH2A-CT (20+20 μM) LLPS by adding different concentrations of ADGRV1-CT (0-60 μM) (see below). 8. For the FRAP assay (Fig. 5H), the authors should add the original movie or the images at different time points. Thank you for the suggestion. We have now added the original images in Fig. S6C. WHRN NPDZ12 and PDZD7 PDZ12 is abbreviated as WHRN and PDZD7, respectively. 9. Full-length Whirlin alone was shown to undergo LLPS. How about full-length Whirlin in this ankle-link complex system? The authors should also comment on it or provide experimental evidence. Thank you for raising this important question for us. Full-length Whirlin alone was reported to undergo LLPS in Lin, L et. al 2020. Therefore, we speculate that the threshold concentration for LLPS of the Whirlin/USH2 protein complex would be lowered if the full-length Whirlin protein is used. We have made the comments in the section of "LLPS in hearing system" in Discussion (Page 9, line 391-395).
10. Where is T110 in the structure? The authors should also show it in Fig. 8A. Thank you for reminding us. We have marked T110 in WHRN NPDZ1 and found it to localize on the αE helix of WHRN NTD.  11. The authors should add more details about how the prediction of the Whirlin/VLGR1 complex structure was performed. Did the authors use Alphafold multimer mode? What sequences were used as input? Such information should be included in the method section. Thank you for providing the essential suggestions on the structure prediction. We have added methodology details on structure prediction in the section of "Alphafold protein structure prediction" in Methods. The amino acid sequence of VLGR1-CT (UniProtKB: Q8VHN7, a.a. 6240-6261) is fused to the C-terminus of Whirlin NTD sequence (UniProtKB: Q9P202, a.a. 33-117) for prediction. And a linker with the amino acid sequence of Glycine-Serine is inserted between VLGR1-CT and Whirlin NTD to provide freedom for Alphafold modeling. The prediction was obtained using the monomeric model of AlphaFold in default parameters.
12. Also, the reliability of the predicted structure should better be validated, preferably by mutagenesis-based binding assays. Thanks for the suggestion. To validate the reliability of the predicted structure of ADGRV1-CT NBM/WHRN NTD, we truncated NBM from ADGRV1-CT for ITC assays. Expectedly, truncating NBM from ADGRV1-CT abolished its binding to WHRN NTD. And compared with the WT protein, ADGRV1-CT △NBM showed decreased binding affinity to WHRN NPDZ12, as the PBM contributed partly to the interaction. We have added the mutagenesis-based ITC assays in Figure S7C (presented below). Reviewer #2 (Remarks to the Author): The manuscript from Wang et al. provides a plausible explanation for how ankle link complexes form in hair cells. Comprehensive ITC and SEC-MALS experiments allowed for the identification of key domains of each of the proteins that bind to each other; the interaction mapping carried out here is very believable and is rigorously done.
Major issues 1. The manuscript badly needs editing from an English-speaking reader. While the erratic grammar does not prevent one from fully understanding the text, it makes it quite a struggle. There are funny turns of phrase, missing articles, plural problems, etc. I could spend hours and hours correcting the grammar but I am unwilling to. Thank you for the suggestion. We have taken extra care in correcting the errors in grammar and polishing the language in the revised manuscript. The American Journal Expert (AJE) helped us with the language editing.
2. The hair-cell localization experiments of Fig. 7 are inadequate. With top-down views of hair bundles at very low resolution, it is impossible to determine where the fluorescent proteins are located. These experiments are critical for the paper-they tie the in vitro biochemistry to hair cell biology-but far better microscopy and more comprehensive experiments are required. We thank the reviewer for noting this issue for us. We consolidated our hair cell localization experiments and updated the images in Figure 7 (presented below for the convenience of viewing). First, to get high-resolution images, we re-injectoporated the expression plasmids, encoding full-length WHRN or WHRN without NTD or PDZ1 domain, into hair cells and examined their localization using a confocal microscope. Then, in order to link the biochemical and structural characterization, human genetic data, and hair-cell experiments together, we also examined the localization of the deafness mutations (EGFP-WHRN-R223H and EGFP-WHRN-A64D) in hair cells. The results reveal that the two-point mutations show a weak effect on the localization of WHRN in the stereocilia (Fig. 7D and E), consistent with our biochemical data and structural analysis. 3. The statement that "[e]lectron densities have been observed at…[the] ankle link region" is not supported by the references used and in fact does not seem to be true. Reference 13 does not include any transmission electron microscopy, required for visualizing the electron dense structures of hair bundles. Reference 22 has TEM of photoreceptors but only SEM of hair cells. Goodyear et al. (2005), a good reference for ankle links, shows some increased electron density in the region of ankle links but that looks extracellular, i.e., because of the density of ADGRV1 and USH2A. I am not aware of any papers clearly showing electron density underneath the ankle links-unlike what is seen with the upper and lower tip link densities. We thank the reviewer for noting this issue for us. We fully agree with the reviewer that TEM Images reported by Goodyear et al. (2005) show some increased electron density in the extracellular region of ankle links. But we also noticed the darker color along and beneath the plasma membrane in the ankle-link region (presented below, panel A). A similar dark density is also reported by McGee, J et al. (2006), providing reliable evidence for our search on the phase separation of the USH2 protein complex (presented below, panel B). We also used electron microscopy to capture the ankle-links of OHC in P2 and observed similar weak enrichment at the ankle-link region, including the extracellular, along, and beneath the plasma membrane. We have now sorted out our references and rephrased this point in our revised version as "Notably, compared to the upper and lower tip-link densities, a weaker electron-dense structure can be also observed at the ankle-link region of stereocilia". This kind of weak density formation is consistent with the temporal assembly property of ankle-link for hair bundle development. The darker densities of the upper and lower tip-link indicate their structural function as rigid anchors, which is similar to the post and pre-synapse density in neurons. It would be interesting to assess the physical properties of different kinds of condensate. For ankle-link condensate, reconstitution of transmembrane proteins that cluster on the supported lipid bilayer can be applied to study these microclusters. 4. Is this really liquid-liquid phase separation? The slow (ADGRV1 and USH2A) or absent (WHRN and PDZD7) FRAP recovery suggests that a solid phase is formed, not liquid. It is important what it is called, as separations into liquid phase or solid phase lead to different biophysical properties. Solid phase is OK, however, for a structural/scaffolding function for the phenomenon you are investigating.
We thank the reviewer for noting this issue. We fully agree with the reviewer that it is important to clarify whether the condensate is more liquid-like or solid-like. So, we combined fluorescence imaging and FRAP assay to describe the phenomenon of phase separation.
First, we monitored the phase droplets under the microscope. The spherical morphology of condensate droplets and their fusion event upon contact suggests the condensates are liquid-like. We have added a sub-panel in Figure S6 (panel B), showing the small droplets gradually fuse into larger ones, reinforcing the conclusion of LLPS. For convenience, we have included the panel below. FRAP analysis is increasingly adopted to demonstrate the mobility and dynamics of molecules within liquid droplets. Molecule exchange within the condensed phase or between the condensed and diluted phases can be captured by FRAP experiments depending on different degrees of bleaching (half bleach or whole bleach). The slow recovery in this study may result from the high photobleaching intensity of the laser, which may engender heat and prevent protein molecules in the surrounding solution from entering the droplets. So, we repeated the FRAP analysis but with different laser intensities for photobleach (100% laser power x 20 times, 100% laser power x 2 times, and 10% laser power x 2 times). The FRAP assay was performed on a Leica SP8 confocal microscope at room temperature with a 63 x oil objective. In each FRAP experiment, only one protein was labeled with Cy3 and added to the mixture at a ratio of 1%. A circular region of interest (ROI) with diameters of 2-3 μm was manually selected for bleach by a 561 nm laser beam. The images were taken in 10 min at 30 s/frame and 30 frames in total. Under different laser intensities, we find that the recovery after photobleaching of both proteins correlates negatively with the intensity of photobleaching.
FRAP assay (A) and PDZD7 (B) under different laser intensities for photobleach (100% laser power x 20 times, 100% laser power x 2 times, and 10% laser power x 2 times). The results were presented as means ± SDs with 3-5 droplets analyzed for each protein. WHRN NPDZ12 and PDZD7 PDZ12 were abbreviated as WHRN and PDZD7, respectively.
We then photobleached USH2A-CT and ADGRV1-CT at the intensity of 10% x 2 times and obtained a new FRAP result with a much higher recovery rate. It is shown that all four proteins appear to recover more quickly than they were photobleached with 100% intensity x20 times (as performed in the original assays). Thus, the newlyconducted FRAP assays demonstrated that the USH2 protein complex in this study really undergoes liquid-liquid phase separation. The new Figure for the FRAP assay is added in Figure 5H. We also summarized some reported recovery rates below. However, due to a lack of detailed descriptions of the laser parameters for photo bleach, we are not able to correlate the recovery rates with the photobleaching intensity, but still, the Other issues 5. Strongly suggest that you use all official protein symbols, especially WHRN instead of Whirlin, USH2A instead of Usherin, and ADGRV1 instead of VLGR1. Consistency in symbols is very important, and I recommend using the protein versions (all caps, no italics) of the official gene symbols. I realize that the names you use are common, but there are many names for the same protein and it's important to use systematic databases. Thank you for the suggestion. Whirlin, Usherin, and VLGR1 have been revised as WHRN, USH2A, and ADGRV1, respectively. 6. Please be thoughtful in reporting significant digits. For example, "66.23 ± 35.17 µM" makes no sense at all, as the number of significant digits indicate precision far, far beyond what the error measurement suggests is a limit. In this example, I would report "66 ± 35 µM" (or even "70 ± 40 µM"). The error value should not have more than two significant digits (not two decimal places), and that should drive reporting of the mean value. Thank you for your kind suggestion. We are sorry for the sloppiness. According to your suggestion, we rephrase the mean value for all the Kd values to take two significant digits. This study provides interesting molecular-level insights into the assembly of the ankle link complex of stereocilia. Through a series of binding and other experiments, they localize relevant interaction domains and characterize the effects of certain residue substitutions. While the results are of interest to the community, more detail regarding methods and results are needed, as noted in the specific comments below.
1. Line 126: "Whirlin NTD bears a high sequence similarity of 60.98%...". Sequence similarities do not have numerical values percentage values; the authors likely mean "sequence identity" and should change the wording in this sentence accordingly. Otherwise, the numerical value should be removed. We thank the reviewer for his/her advice and we have now modified the text: "The amino acid sequence of WHRN NTD is highly similar to Harmonin NTD…" (Page 3, Line 115-116) 2. In Figure 6E, the authors label the right-most panel with the "VLGR1-CT NBM/Whirlin NTD". Although the figure legend notes that this is a model, to make this more clear to readers that are viewing the figure, the authors should clearly note that it is a model in the panel title, e.g.: (AlphaFold) "Model of VLGR1-CT…". We have modified the panel title as "Model of ADGRV1-CT NBM/WHRN NTD" in the revised paper.
3. The description of the AlphaFold modeling procedure (lines 563-570) requires more information. A fusion is described in this section, but it notes Usherin-CT rather than VLGR1-CT, which does not seem to be in accordance with the reported model. The authors should clearly note the specific residue ranges and proteins used as input to AlphaFold, and for the fusion they should note whether a linker was used (which would be expected, to permit degrees of freedom for AlphaFold modeling), and if a linker was used, the authors should describe its length and constitution (e.g. poly-Glycine). Additionally, they should note whether default AlphaFold parameters were used, and whether AlphaFold-Multimer or AlphaFold (monomeric model) were used to model the complex. Thank you for the suggestions above. VLGR1-CT is used for sequence fusion in the model prediction, we are extremely sorry for the typo mistake in the draft. And we have added the missing methodology information in the section of "Alphafold protein structure prediction" in Methods. The amino acid sequence of VLGR1-CT (UniProtKB: Q8VHN7, a.a. 6240-6261) is fused to the C-terminus of Whirlin NTD sequence (UniProtKB: Q9P202, a.a. 33-117) for prediction. And a linker with the amino acid sequence of Glycine-Serine is inserted between VLGR1-CT and Whirlin NTD to provide freedom for Alphafold modeling. The prediction was obtained using the monomeric model of AlphaFold in default parameters.
4. The authors note that pLDDT confidence scores were used to select the AlphaFold model, but they do not seem to report the actual pLDDT score of the model that they report. Both pLDDT and pTM AlphaFold model confidence scores should be noted by the authors in the Results and/or Figure 6 legend, to provide readers with a better understanding of the model's confidence. Sorry for the missing information. In the revised manuscript, we added the pLDDT score in both Methods and Result sections. The predicted structure was generated by the monomer mode with default parameters which only provide pLDDT but not pTM value. The pLDDT score is 87.86 for ADGRV1-CT NBM/WHRN NTD.
We also conducted a biochemistry assay to validate the reliability of the predicted structure of ADGRV1-CT NBM/WHRN NTD. We truncated NBM from ADGRV1-CT for ITC assays. Truncating NBM from ADGRV1-CT abolished its binding to WHRN NTD. And compared with the WT protein, ADGRV1-CT △NBM showed decreased binding affinity to WHRN NPDZ12, as the PBM contributed partly to the interaction. We have added the mutagenesis-based ITC assays in Figure S7C (presented below). 5. The representation of the Whirlin-NTD cartoon in Figure 8A (right-hand part) seems clipped in the superposition with the Harmonin-NTD, resulting in multiple apparent breaks in the helix. The authors should try to adjust this representation, as the helix breaks are distracting and potentially misleading. Thank you for reminding us. We have adjusted the Figure to avoid breaks.